Human iPSC-Derived Cardiac Myocytes: Toward an In Vitro Model of Cardiac Physiology

University of Minnesota Ph.D. dissertation. May 2017. Major: Integrative Biology and Physiology. Advisor: Joseph Metzger. 1 computer file (PDF); vii, 187 pages.Cardiovascular Disease is a growing public health issue in the modern world, with a high incidence rate that continues to increase, and poor mortality rates. Recent technological advances have made it possible to efficiently derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). These have been seen as a model for human heart disease, as well as a potential source for cellular transplantation into failing diseased heart tissue. Many laboratories have devoted substantial effort to examining the functional properties of hiPSC-CMs, including electrophysiology, intracellular calcium handling, and gene/protein expression and force. In the first part of this thesis, we utilize traction force microscopy (TFM) to determine the maximum force production of isolated hiPSC-CMs under varied culture and assay conditions. We elucidate here the relationship between cell morphology and force production, and find a significant relationship between cell size and force. HiPSC-CMs developing in culture for two weeks produce significantly less force than cells cultured from one to three months and hiPSC-CMs cultured for three months resemble the cell morphology of neonatal rat ventricular myocytes. Unexpectedly, hiPSC-CMs produce less force when assayed on increasingly stiff substrates, and generate less strain energy. Finally, hiPSC-CMs cultured in conditions of physiologic calcium concentrations are larger and produce more force than cells cultured in standard media. In the second part of this thesis, we address the concept of immaturity in hiPSC-CMs, and attempt to accelerate maturation. We use genome editing to engineer hiPSC-CMs that contain an inducible gene expression cassette, in order to overexpress two proteins associated with maturity: SERCA2a and cardiac troponin I (cTnI). We find that we are able to overexpress both proteins in differentiated hiPSC-CMs after two weeks of treatment with doxycycline. SERCA2a-overexpressing cells showed significant alterations in physiologic function, including increased chronotropy and decreased time to peak in calcium transients following treatment with isoproterenol, a β-adrenergic agonist. Furthermore, using an impedance-measuring system to track contractility kinetics, we found that SERCA2a-overexpressing cells had shortened time to peak and time to baseline after gene induction, with continued response to isoproterenol. As a sign of maturation, SERCA cells also expressed increased cTnI, a key marker of maturity. Using RNAseq, we found that cTnI-overexpressing cells had marked, global changes in their gene expression profile. Key findings include upregulation of genes associated with cardiac contractility and development, such as cardiac myomesin and tropomyosin and ryanodine receptor, and downregulation of genes associated with pacemaker and ventricular cell types, such as HCN and GREM2, and genes associated with skeletal myocytes, such as skeletal muscle actin. Overall, our findings show that hiPSC-CMs have physiologic function similar to that of immature cardiac myocytes, but that we are able to induce maturation by overexpression of genes associated with maturity

Factors affecting the evolution of mimicry

Ph. D. Thesis.Mimicry, where an undefended species resembles a defended species (Batesian mimicry) or where two or more defended species resemble one another (Müllerian mimicry) is one of the most fascinating examples of natural selection in nature. However, even after more than 150 years of research, there are still outstanding questions. One of the biggest of these is: Why do some mimics resemble their models more closely than others? Several hypotheses have been proposed to explain this, yet few have been tested experimentally. To do this, I collected images of museum specimens of real-life model-mimic pairs using a hyperspectral scanner. I then analysed these images to measure the similarity of model-mimic pairs to a potential avian predator. I then investigated how these measures were affected by three factors which have previously been suggested to influence mimetic similarity: the palatability of the mimic, the climate of the area where the mimic is found and the size of the mimic. None of these factors had a significant effect on any measures of similarity. I then performed two behavioural experiments using domestic chicks (Gallus gallus domesticus) as predators of artificial prey, in order to determine whether the nutritional value of prey influences the degree to which predators discriminate between models and Batesian mimics. I found no direct evidence to support this hypothesis. When taken together, the results of my experiments highlight how much there is still to learn about mimicry as well as the need to test many of the hypotheses surrounding it

Hermit crabs (<i>Pagurus bernhardus</i>) use visual contrast in self- assessment of camouflage

Animals can make use of camouflage to reduce the likelihood of visual detection or recognition and thus improve their chances of survival. Background matching, where body colouration is closely matched to the surrounding substrate, is one form of camouflage. Hermit crabs have the opportunity to choose their camouflage independently of body colouration as they inhabit empty gastropod shells, making them ideal to study their choice of camouflage. We used 3D-printed artificial shells of varying contrasts against a grey substrate to test whether hermit crabs prefer shells that they perceive as less conspicuous. Contrast-minimising shells were chosen for Weber contrasts stronger than −0.5. However, in looming experiments, animals responded to contrasts as weak as −0.2, indicating that while they can detect differences between shells and the background, they are only motivated to move into those shells when the alternatives contrast strongly. This suggests a trade-off between camouflage and vulnerability introduced by switching shells

Recruiting women with ductal carcinoma in situ to a randomised controlled trial: lessons from the LORIS study

BackgroundThe LOw RISk DCIS (LORIS) study was set up to compare conventional surgical treatment with active monitoring in women with ductal carcinoma in situ (DCIS). Recruitment to trials with a surveillance arm is known to be challenging, so strategies to maximise patient recruitment, aimed at both patients and recruiting centres, were implemented.MethodsWomen aged ≥ 46 years with a histologically confirmed diagnosis of non-high-grade DCIS were eligible for 1:1 randomisation to either surgery or active monitoring. Prior to randomisation, all eligible women were invited to complete: (1) the Clinical Trials Questionnaire (CTQ) examining reasons for or against participation, and (2) interviews exploring in depth opinions about the study information sheets and film. Women agreeing to randomisation completed validated questionnaires assessing health status, physical and mental health, and anxiety levels. Hospital site staff were invited to communication workshops and refresher site initiation visits to support recruitment. Their perspectives on LORIS recruitment were collected via surveys and interviews.ResultsEighty percent (181/227) of eligible women agreed to be randomised. Over 40% of participants had high anxiety levels at baseline. On the CTQ, the most frequent most important reasons for accepting randomisation were altruism and belief that the trial offered the best treatment, whilst worries about randomisation and the influences of others were the most frequent most important reasons for declining. Most women found the study information provided clear and useful. Communication workshops for site staff improved knowledge and confidence but only about half said they themselves would join LORIS if eligible. The most common recruitment barriers identified by staff were low numbers of eligible patients and patient preference.ConclusionsRecruitment to LORIS was challenging despite strategies aimed at both patients and site staff. Ensuring that recruiting staff support the study could improve recruitment in similar future trials

Stellar, brown dwarf and multiple star properties from a radiation hydrodynamical simulation of star cluster formation

We report the statistical properties of stars, brown dwarfs and multiple systems obtained from the largest radiation hydrodynamical simulation of star cluster formation to date that resolves masses down to the opacity limit for fragmentation (a few Jupiter masses). The initial conditions are identical to those of previous barotropic calculations published by Bate, but this time the calculation is performed using a realistic equation of state and radiation hydrodynamics. The calculation uses sink particles to model 183 stars and brown dwarfs, including 28 binaries and 12 higher-order multiple systems, the properties of which are compared the results from observational surveys. We find that the radiation hydrodynamical/sink particle simulation reproduces many observed stellar properties very well. In particular, whereas using a barotropic equation of state produces more brown dwarfs than stars, the inclusion of radiative feedback results in a stellar mass function and a ratio of brown dwarfs to stars in good agreement with observations of Galactic star-forming regions. In addition, many of the other statistical properties of the stars and brown dwarfs are in reasonable agreement with observations, including multiplicity as a function of primary mass, the frequency of very-low-mass binaries, and general trends for the mass ratio and separation distributions of binaries. We also examine the velocity dispersion of the stars, the distributions of disc truncation radii due to dynamical interactions, and coplanarity of orbits and sink particle spins in multiple systems. Overall, the calculation produces a cluster of stars whose statistical properties are difficult to distinguish from observed systems, implying that gravity, hydrodynamics, and radiative feedback are the primary ingredients for determining the origin of the statistical properties of low-mass stars.Comment: Accepted for publication in MNRAS, 33 pages, 23 figures. Animations can be found at http://www.astro.ex.ac.uk/people/mbate

Investigation of human iPSC-derived cardiac myocyte functional maturation by single cell traction force microscopy.

Recent advances have made it possible to readily derive cardiac myocytes from human induced pluripotent stem cells (hiPSC-CMs). HiPSC-CMs represent a valuable new experimental model for studying human cardiac muscle physiology and disease. Many laboratories have devoted substantial effort to examining the functional properties of isolated hiPSC-CMs, but to date, force production has not been adequately characterized. Here, we utilized traction force microscopy (TFM) with micro-patterning cell printing to investigate the maximum force production of isolated single hiPSC-CMs under varied culture and assay conditions. We examined the role of length of differentiation in culture and the effects of varied extracellular calcium concentration in the culture media on the maturation of hiPSC-CMs. Results show that hiPSC-CMs developing in culture for two weeks produced significantly less force than cells cultured from one to three months, with hiPSC-CMs cultured for three months resembling the cell morphology and function of neonatal rat ventricular myocytes in terms of size, dimensions, and force production. Furthermore, hiPSC-CMs cultured long term in conditions of physiologic calcium concentrations were larger and produced more force than hiPSC-CMs cultured in standard media with sub-physiological calcium. We also examined relationships between cell morphology, substrate stiffness and force production. Results showed a significant relationship between cell area and force. Implementing directed modifications of substrate stiffness, by varying stiffness from embryonic-like to adult myocardium-like, hiPSC-CMs produced maximal forces on substrates with a lower modulus and significantly less force when assayed on increasingly stiff adult myocardium-like substrates. Calculated strain energy measurements paralleled these findings. Collectively, these findings further establish single cell TFM as a valuable approach to illuminate the quantitative physiological maturation of force in hiPSC-CMs

A computational neuroscience framework for quantifying warning signals

1. Animal warning signals show remarkable diversity, yet subjectively appear to share certain visual features that make defended prey stand out and look different from more cryptic palatable species. For example, many (but far from all) warning signals involve high contrast elements, such as stripes and spots, and often involve the colours yellow and red. How exactly do aposematic species differ from non-aposematic ones in the eyes (and brains) of their predators?2. Here, we develop a novel computational modelling approach, to quantify prey warning signals and establish what visual features they share. First, we develop a model visual system, made of artificial neurons with realistic receptive fields, to provide a quantitative estimate of the neural activity in the first stages of the visual system of a predator in response to a pattern. The system can be tailored to specific species. Second, we build a novel model that defines a ‘neural signature’, comprising quantitative metrics that measure the strength of stimulation of the population of neurons in response to patterns. This framework allows us to test how individual patterns stimulate the model predator visual system.3. For the predator–prey system of birds foraging on lepidopteran prey, we compared the strength of stimulation of a modelled avian visual system in response to a novel database of hyperspectral images of aposematic and undefended butterflies and moths. Warning signals generate significantly stronger activity in the model visual system, setting them apart from the patterns of undefended species. The activity was also very different from that seen in response to natural scenes. Therefore, to their predators, lepidopteran warning patterns are distinct from their non-defended counterparts and stand out against a range of natural backgrounds.4. For the first time, we present an objective and quantitative definition of warning signals based on how the pattern generates population activity in a neural model of the brain of the receiver. This opens new perspectives for understanding and testing how warning signals have evolved, and, more generally, how sensory systems constrain signal design